1. Technical Field of the Invention
This invention relates generally to cytoenzymology, and more particularly to methods and reagents used in cytoenzymology.
2. Discussion of the Background Art
Apoptosis, or programmed cell death, is a process that involves the activation of a genetic program when cells are no longer needed or have become seriously damaged. This process, occurring in most cells from higher eukaryotes, is necessary for normal development and maintenance of homeostasis. It is a major defense mechanism of the body, getting rid of unwanted and possibly dangerous cells such as virus-infected cells, tumor cells, and self-reactive lymphocytes. Apoptosis probably exists as a safeguard to prevent damaged cells from reproducing. If the damaged cells remained, they may ultimately be detrimental to the organism.
Apoptosis involves a cascade of specific biochemical events. Regulation of apoptosis involves a large number of genes. These can be classified into three general categories. The first is made up of genes whose primary function is to suppress apoptosis. This group includes some members of the bcl-2 family. Other members of the bcl-2 family can promote apoptosis. The second group includes the intermediate genes upstream such as Fas/Fas ligand, myc, p53, and WAF1. The last group includes genes that act as effectors of apoptosis. An example is the interleukin-1.beta. converting enzyme (ICE) family of genes.
Fas is a cell surface protein that triggers apoptosis in a variety of cell types. The Fas death pathway can be triggered by either anti-Fas monoclonal antibody or by cell-associated Fas ligand. This protein is identical to the CD95 protein. CD95 is involved in regulation of tissue development and homeostasis. Cloning of Fas and APO-1 cDNA has demonstrated that these two genes are identical. The Fas antigen is a cell surface protein that belongs to the tumor necrosis factor/nerve growth factor receptor family. The mechanism by which Fas triggers cell death is not completely understood. It appears to require multivalent cross-linking of the receptor. This inhibits RNA and protein synthesis in certain cell types. The amino acid sequences that take part in cell death have been mapped in Fas and tumor necrosis factor receptors. This area is called the "death domain" and is essential for the initiation of apoptosis, which may happen through an interaction with other intracellular proteins.
Many cysteine proteases have been identified in mammals. Interleukin-1.beta.-converting enzyme (ICE) is a cysteine protease whose activity is increased in apoptosis. Several homologues of ICE have been identified, including CPP32- and Ich-1-like proteases. Specific inhibitors of ICE-like proteases can inhibit apoptosis. This indicates there is a requirement for specific degradation by proteases in mammalian apoptosis. The ICE family of cysteine proteases has an indispensable role in the regulation of apoptosis.
It appears that the ICE family of proteases process themselves and each other by proteolytically cleaving a "pre" enzyme into the active form. The ICE family of proteases is generically referred to as caspase enzymes. Alnemri, et al., Cell, Volume 87, page 171, 1996. The "c" is intended to reflect a cysteine protease mechanism and "aspase" refers to their ability to cleave after aspartic acid, the most distinctive catalytic feature of this protease family.
Enari, et al, Nature 380, pages 723-726 (1996) have suggested that apoptotic events may sequentially activate ICE and CPP32-like proteases in Fas-mediated apoptosis. Enari used specific inhibitors of ICE and CPP32 to show that CPP32-inhibitor only inhibited CPP32 activity. ICE-inhibitor, on the other hand, inhibited both ICE and CPP32 activity. This indicates that the production of CPP-32-like activity during Fas-mediated apoptosis depends on the previous presence of ICE-like activity. This experiment was performed in a cytosol of approximately a billion human cells.
Cell death can occur by either necrosis or apoptosis. Necrosis, which is not genetically controlled, is usually the result of physical injury. Apoptosis is genetically controlled and is the deliberate cellular response to specific environmental and developmental stimuli. The signs of necrosis and apoptosis are different. Necrosis involves the destruction of cytoplasmic organelles and a loss of plasma membrane integrity. Cells undergoing apoptosis exhibit cell shrinkage, membrane blebbing, chromatin condensation and fragmentation. After the DNA damage in the caspase enzyme pathway, there are a series of events which occur that involve calcium activation and calpain enzymes which further leads to other cellular changes and regulation of cytoplasmic enzymes.
A major difference between necrosis and apoptosis in vivo is the complete elimination of the apoptotic cell before an inflammatory response is seen. Necrosis usually causes inflammation. Thus, apoptosis can be thought of as a clean and neat process where cells are killed with minimal damage to surrounding cells or tissue.
One test to study proteases is to provide a substrate that is recognized by the enzyme, with a fluorescent compound which will undergo a detectable change when the substrate, or "leaving group", is cleaved from the compound by the enzyme. Mangel et al., U.S. Pat. Nos. 4,557,862 and 4,640,893, disclose Rhodamine 110-based derivatives as fluorogenic substrates for proteinases. These compounds have the general formula: EQU (Cbz-peptide-NH).sub.2 -Rhodamine 110
where the peptide includes known amino acids or amino acid derivatives, and "Cbz" refers to the blocking group benzyloxycarbonyl. When the amino groups of Rhodamine 110 are blocked the compound is "quenched", and is relatively colorless and non-fluorescent. Cleavage of one of the peptides from the non-fluorescent bisamide substrate results in a 3500-fold increase in fluorescence intensity.
Other substrates for conducting cytoenzymological studies are sold by Kamiya Biomedical Company, Seattle Washington and have the formula (Z-Asp Glu Val Asp-AFC), (Z-Ala Ala Asp-AFC) and (Z-Tyr-Val-Ala-Asp-AFC), wherein AFC is 7-amino4 trifluoromethyl coumarin and Asp Glu Val Asp, Ala Ala Asp and Tyr-Val-Ala-Asp are abbreviations for amino acids as later defined in this specification. However, these reagents either bind to the external cell membrane or exhibits solubility problems so that they fail to measure intracellular enzymes. In addition, courmarin excite and emits in the ultraviolet light range which is known to cause DNA damage which is detrimental to cell viability.
Prior method and reagents have failed to provide an effective test to monitor the efficacy of a drug or the progression of a disease through the use of an intracellular analysis of apoptosis. Moreover, the present invention provides an additional feature of distinguishing between apoptosis and necrosis by using enzymes other than the ICE family of caspase enzymes.